Methods for determining whether a compound alters the amount of at least one αβ (X-41) peptide and the amount of either total αβ or at least one αβ (X-40) peptide produced by a non-human mammal

ABSTRACT

This invention provides methods of screening compounds for their ability to alter the production of Aβ(x≧41) alone or in combination with Aβ(x≦40). The methods involve administering compounds to cells, specifically measuring the amounts of Aβ(x≦40) and Aβ(x≧41) produced by the cells, and comparing these amounts to that produced by the cells without administration of the compounds.

This application is a divisional of U.S. application Ser. No. 08/665,649filed Jun. 18, 1996 which is now U.S. Pat. No. 6,610,493.

This invention was made in part with Government support under grant no.2 P50 AG05134 awarded by the National Institutes of Health. TheGovernment may have rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to neurology and, moreparticularly, to assays, such as immunoassays, for screening forcompounds that specifically alter the production of various isoforms ofAβ.

Alzheimer's disease (AD) is a degenerative brain disorder characterizedclinically by progressive loss of memory, cognition, reasoning, judgmentand emotional stability that gradually leads to profound mentaldeterioration and ultimately death. AD is a very common cause ofprogressive mental failure (dementia) in aged humans and is believed torepresent the fourth most common medical cause of death in the UnitedStates. AD has been observed in all races and ethnic groups worldwideand presents a major present and future public health problem. Thedisease is currently estimated to affect about two to three millionindividuals in the United States alone. AD is at present incurable. Notreatment that effectively prevents AD or reverses its symptoms orcourse is currently known.

The brains of individuals with AD exhibit characteristic lesions termedsenile plaques and neurofibrillary tangles. Large numbers of theselesions are generally found in patients with AD in several areas of thehuman brain important for memory and cognitive function. Smaller numbersof these lesions in a more restricted anatomical distribution aresometimes found in the brains of aged humans who do not have clinicalAD. Senile plaques and vascular amyloid deposits (amyloid angiopathy)also characterize the brains of individuals beyond a certain age withTrisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage withAmyloidosis of the Dutch-Type (HCHWA-D). The principal chemicalconstituent of the senile plaques and vascular amyloid depositscharacteristic of AD and the other disorders mentioned above is aprotein designated the amyloid-β peptide (Aβ) or sometimes βAP, AβP orβ/A4. Aβ was first purified and a partial amino acid sequence reportedin Glenner and Wong (1984) Biochem. Biophys. Res. Commun.120:885-890.The isolation procedure and the sequence data for the first 28 aminoacids are described in U.S. Pat. No. 4,666,829. Forms of Aβ having aminoacids beyond number 40 were first reported by Kang et al. (1987) Nature325:733-736.

Roher et al. (1993) Proc. Natl. Acad. Sci. USA 90:10836-840 showed thatAβ(1-42) is the major constituent in neuritic plaques, includingsignificant amounts of isomerized and racemized aspartyl residues astheir NH₂-termini. The authors also reported that Aβ(17-42) (p3(42))predominates in diffuse (early) plaques, whereas Aβ(1-40) is the majorconstituent in the meningeal vessel deposits, comprising 60% of thetotal Aβ in those vessels. Iwatsubo et al. (1994) Neuron 13:45-53 showedthat Aβ42(43)-containing senile plaques are the major species of senileplaques in sporadic AD brains. Iwatsubo et al. (1995) Annals ofNeurology 37:294-299 and Lemere et al. (1996) Neurobiology of Disease3:16-32 reported that Aβ42(43) is the major constituent of senileplaques in Down's syndrome brains and is the initially deposited Aβspecies in the development of AD-type neuropathological legions in thesepatients. In addition, Gravina et al., (1995) J. Biol. Chem.270:7013-7016 reported both biochemical and immunocytochemical evidencethat Aβ42(43) peptides were the most abundant constituents of senileplaques in AD brains and exceeded the amounts of Aβ40 peptides in suchplaques.

Molecular biological and protein chemical analyses conducted during thelast several years have shown that Aβ is a small fragment of a muchlarger precursor protein, referred to as the β-amyloid precursor protein(APP), that is normally produced by cells in many tissues of variousanimals, including humans. Knowledge of the structure of the geneencoding APP has demonstrated that Aβ arises as a peptide fragment thatis cleaved from the carboxy-terminal end of APP by as-yet-unknownenzymes (proteases). The precise biochemical mechanism by which the Aβfragment is cleaved from APP and subsequently deposited as amyloidplaques in the cerebral tissue and in the walls of cerebral andmeningeal blood vessels is currently unknown. Importantly, Haass et al.(Nature 359:322-325) and Seubert et al. ((1992) Nature 359:325-327)discovered that essentially all cells expressing the APP gene normallysecrete an array of Aβ peptides, and these peptides can readily bedetected and assayed in cell culture fluid (conditioned media) and humanbiological fluids such as plasma and cerebrospinal fluid. It hassubsequently been shown that these fluids contain both the more abundantAβ40-ending peptides and the less abundant Aβ42(43)-ending peptides(Dovey et al. (1993) Neuroreport 4:1039-1042 and Vigo-Pelfrey et al.(1993) J. Neurochem. 61:1965-68)

Several lines of evidence indicate that progressive cerebral depositionof Aβ plays a seminal role in the pathogenesis of AD and can precedecognitive symptoms by years or decades (for review, see Schenk (1995) J.Med. Chem. 38:4141-4154, Selkoe (1994) J. Neuropath. and Exp. Neurol.53:438-447 and Selkoe (1991) Neuron 6:487). One of the most importantlines of evidence is the discovery in 1991 that missense DNA mutationsin the APP gene at amino acid 717 of the 770-amino acid isoform of APPcan be found in affected members but not unaffected members of severalfamilies with a genetically determined (familial) form of AD (Goate etal. (1991) Nature 349:704-706; Chartier Harlan et al. (1991) Nature353:844-846; and Murrell et al. (1991) Science 254:97-99). Suzuki et al.(1994) “An increased percentage of long amyloid β-protein secreted byfamilial amyloid β-protein precursor (βAPP717) mutants,” Science264:1336-1340 subsequently showed that, compared to normal individuals,the 717 mutation causes a higher relative production of the Aβ(1-42)peptide. In addition, a double mutation changing lysine⁶⁷⁰-methionine⁶⁷¹to asparagine⁶⁷⁰-leucine⁶⁷¹ (with reference to the 770 isoform of APP)was reported in a Swedish family with familial AD in 1992 (Mullan et al.(1992) Nature Genet 1:345-347) and is referred to as the Swedish APPvariant.

Genetic linkage analyses have demonstrated that the aforementionedmutations are the specific molecular cause of AD in the members of suchfamilies that carry these mutant APP genes. In addition, a mutation atamino acid 693 of the 770-amino acid isoform of APP has been identifiedas the cause of the Aβ deposition disease, Hereditary CerebralHemorrhage With Amyloidosis Dutch type (HCHWA-D), and a mutation fromalanine to glycine at amino acid 692 appears to cause the phenotype ofAD in some family members and the phenotype of HCHWA-D in others. Thediscovery of these APP mutations in genetically based cases of AD arguesthat genetic alteration of APP and subsequent deposition of its Aβfragment can cause AD.

Recently, evidence has accumulated suggesting that Aβ(42) plays the keyrole in the process of senile plaque formation in AD. First, in vitrodata demonstrate that Aβ(42) accelerates the formation of Aβ fibrils(and thus senile plaques) by a nucleation dependent mechanism (Jarrettet al. (1993) Biochemistry 32:4693-4697). Second, while accounting for≦10% of total Aβ secreted from cells (roughly 90% is Aβ(40) (Dovey etal. (1993) Neuroreport 4:1039-1042; Asami-Odaka et al. (1995) “Longamyloid β-protein secreted from wild-type human neuroblastoma IMR-32cells.” Biochemistry 34:10272-10278), Aβ(42) is the major plaquecomponent (Kang et al. (1987) Nature 325:733-736; Iwatsubo et al. (1994)Neuron 13:45-53; Iwatsubo et al. (1995) Ann. Neurol. 37:294-299; Gravinaet al. (1995) J. Biol. Chem. 270:7013-7016; Lemere et al. (1996)Neurobiology of Disease 3:16-32). Furthermore, all 3 early onsetfamilial AD genes identified to date have been shown to lead to anincrease in cellular secretion of Aβ(42). Only the Swedish APP missensemutation increases the secretion of both Aβ(40) and Aβ(42) peptides(Dovey et al. (1993) Neuroreport 4:1039-1042, whereas the APP717mutations and the presenilin mutations appear not to increase Aβ(40)peptides (Suzuki et al., (1994) Science 264:1336-1340; Scheuner et al.(1995) Neurosci. Abstracts in press). Thus, the longer Aβ(42) peptideappears to be a prime target for therapeutic intervention. However, noneof the proteases involved in the major steps of APP processing have beendefinitively identified, including γ-secretase, the protease whichgenerates the C-terminus of Aβ. It has generally been assumed that thesame protease(s) generate both Aβ(40) and A(42) and it has been shownthat both forms share a common secretory mechanism which involves acidicintracellular compartments such as the late Golgi or early endosomes(Koo and Squazzo (1994) J. Biol. Chem. 269:17386-17389; Asami-Odaka etal. (1995) Biochemistry 34:10272-10278). Recently, Higaki et al. ((1995)Neuron, 14:651-659) have shown that the Calpain inhibitor, MDL 28170,inhibits the production of both total Aβ and total p3 and leads to anaccumulation of their respective 12 kDa and 10 kDa APP precursorfragments in treated cells. These data suggest that the compounddirectly inhibits at least some form of γ-secretase although no data areprovided as to what specific form of Aβ and p3 are affected.

Despite the progress which has been made in understanding the underlyingmechanisms of AD, there remains a need for assays to identify candidatecompounds for preventing or treating the disease.

SUMMARY OF THE INVENTION

According to current theory, the processing of APP is believed toinvolve several specific cleavages by proteases. The enzyme that cleavesAPP between amino acids 671/672 (referring to the βAPP₇₇₀ isoform) iscalled β-secretase. The enzyme that cleaves between amino acids 687/688of APP (16/17 of Aβ) is called α-secretase. Until now it was believedthat cleavage of APP that yielded Aβ(40) and Aβ(42) was carried out by asingle enzyme called γ-secretase. However, we have discovered that acompound can inhibit the production of Aβ(40) but not Aβ(42). Inparticular, we have discovered that compounds, thought to inhibit theproduction Aβ in general, actually inhibit production of Aβ(40) but notAβ(42). This indicates that multiple γ-secretase mechanisms are at workwhich can be pharmacologically dissociated.

Because Aβ(42) is the major component of β-amyloid plaques and initiatesamyloid plaque formation in AD patients, it is important to have toolsto screen compounds to identify those that specifically inhibit theproduction of Aβ(42) and Aβ(40), either simultaneously or separately.The current invention provides such assays.

This invention provides methods for determining whether a compoundalters the amount of at least one Aβ(x-≧41) peptide produced by a cell.The methods involve administering the compound to a culture comprisingthe cell; measuring the amount of the Aβ(x-≧41) peptide, specifically,in a sample from the culture; and determining whether the measuredamount is different than the amount expected in a sample from a culturecomprising the cell to which no compound has been administered. Adifference between the measured amount and the expected amount indicatesthat the compound alters the amount of an Aβ(x-≧41) peptide produced bythe cell.

In another aspect, this invention provides methods for determiningwhether a compound alters the amount of at least one Aβ(x-≧41) peptideproduced by a cell and alters the amount of either total Aβ or at leastone Aβ(x-≦40) peptide produced by the cell. The methods involveadministering the compound to a culture comprising the cell; measuringthe amount of the Aβ(x-≧41) peptide, specifically, in a sample from theculture; measuring the amount of total Aβ or the Aβ(x-≦40) peptide,specifically, in a sample from the culture; and determining whether themeasured amounts are different than the amounts expected in a samplefrom a culture comprising the cell to which no compound has beenadministered. Differences between the measured amounts and the expectedamounts indicate that the compound alters the amount of the Aβ(x-≧41)peptide by a cell and/or the amount of total Aβ or the Aβ(x-≦40) peptideby the cell.

In one embodiment, the amount of the Aβ peptides are measured byimmunoassay and, in particular, sandwich immunoassay comprising capturebinding substances bound to a solid phase and a labeled detectionbinding substance.

In sandwich assays for determining the amount of at least one Aβ(x-≧41)peptide, the capture antibody preferably is specific for Aβ(x-≧41)peptides, e.g., raised against peptideNH₂-Cys-NH—CH₂—(CH₂)₅—CO-GLMVGGVVIA-COOH (SEQ ID NO: 4). The detectionbinding substance in this assay can be an antibody specific for Aβpeptides whose amino-terminal amino acid is no. 1 of Aβ, or can bespecific for an epitope within the junction region of Aβ. In anotherembodiment the capture binding substance for measuring the amount of atleast one of Aβ(x-≧41) peptide is specific for an epitope within thejunction region of Aβ and the detection binding substance is an antibodyspecific for Aβ(x-≧41).

In sandwich assays for determining the amount of at least one Aβ(x-≦40)peptide, the capture binding substance preferably is an antibodyspecific for Aβ(x-≦40) peptides, e.g., raised against the peptideNH₂-Cys-NH—CH₂—(CH₂)₅—CO-GLMVGGVV-COOH (SEQ ID NO: 5). The labeleddetection binding substance can be an antibody specific for the Aβpeptides whose amino-terminal amino acid is no. 1 of Aβ or an antibodyspecific for an epitope within the junction region of Aβ. In anotherembodiment the capture binding substance for measuring the amount of atleast one of Aβ(x-≦40) peptide is specific for an epitope within thejunction region of Aβ and the detection binding substance is an antibodyspecific for Aβ(x-≦40).

In sandwich assays for determining the amount of total Aβ, the capturebinding substance preferably is an antibody specific for an epitopewithin the junction region of Aβ. The detection binding substancepreferably is specific for Aβ peptides whose amino-terminal amino acidis no. 1 of Aβ.

In another embodiment of an immunoassay, the step of measuring theamount of the Aβ(x-≧41) peptide, total Aβ or the Aβ(x-≦40) peptide in asample from the culture comprises: pulsing the culture with aradioactive label for protein; chasing the culture without a radioactivelabel; administering the compound to the cell during the chase period;contacting a sample from the culture with a binding substance specificfor Aβ(x-≧41) peptides; contacting a sample from the culture with abinding substance specific for total Aβ or Aβ(x-≦40) peptide; anddetermining the amount of radioactive label attached to the bindingsubstances.

In other embodiments of the methods the culture comprises primary humanneurons, primary neurons from a transgenic PDAPP mouse (i.e., atransgenic mouse whose cells harbor a PDAPP construct), a 293 humankidney cell line, a human neuroglioma cell line, a human HeLa cell line,a primary endothelial cell line, a primary human fibroblast line, aprimary lymphoblast line, human mixed brain cells, or a Chinese hamsterovary (CHO) cell line. In one embodiment the cell is a host celltransfected with a recombinant expression vector encoding a human APP,e.g., a Hardy mutation such as V717F or the Swedish mutant; causing thecell to overproduce Aβ(x-≧41) peptides. In another aspect, the methodsfurther comprise the step of determining whether the compound is toxicto the cell.

In another aspect this invention provides kits for specificallydetecting at least one Aβ(x-≧41) peptide and at least one Aβ(x-≦40)peptide in a sample. The kits include a binding substance specific forAβ(x-≧41) peptides; and a binding substance specific for Aβ(x-≦40)peptides.

In another aspect this invention provides kits for specificallydetecting at least one Aβ(x-≧41) peptide and either total Aβ or at leastone Aβ(x-≦40) peptide in a sample in a sandwich immunoassay. The kitsinclude at least two different binding substances for measuring theamount of Aβ(x-≧41) peptide; and at least two different bindingsubstances for measuring the amount of total Aβ or Aβ(x-≦40) peptides.

In another aspect this invention provides methods for determiningwhether a compound alters the amount of at least one Aβ(x-≧41) peptideproduced by a non-human mammal and alters the amount of either total Aβor at least one Aβ(x-≦40) peptide produced in the non-human mammal. Themethods involve measuring a first amount of the Aβ(x-≧41) peptide in asample from a non-human animal used as a model of Alzheimer's disease;measuring a first amount of total Aβ or the Aβ(x-≦40) peptide in asample from the non-human animal; administering the compound to thenon-human animal; measuring a second amount of the Aβ(x-≧41) peptide ina sample from the non-human animal; measuring a second amount of totalAβ or the Aβ(x-≦40) peptide in a sample from the non-human animal; andcomparing the first amounts with the second amounts. The comparisonindicates whether the compound increases, decreases, or leaves unchangedthe amount of the Aβ(x-≧41) peptide and increases, decreases, or leavesunchanged the amount of the Aβ(x-≦40) peptide. In certain embodimentsthe non-human animal is a rodent, in particular, a mouse. The non-humananimal can harbor a copy of an expressible transgene sequence whichencodes a Hardy mutation (e.g., V717F) or the Swedish mutation of humanβ-amyloid precursor protein (APP).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Shows standard curves of Aβ(1-40) and Aβ(1-42) with 2G3 as acapture antibody.

FIG. 2. Shows standard curves of Aβ(1-40) and Aβ(1-42) with 21F12 as acapture antibody.

FIG. 3. Shows that compound MDL 28170 influences βAPP metabolism. (0)untreated K695sw cells; (200) K695sw cells treated with 200 μM MDL 28170during the chase period. Lanes 1,2: aliquot of total chase media rundirectly on the gel. Lanes 3,4: antibody 1736 immunoprecipitation ofchase media. Lanes 5,6: antibody 192sw immunoprecipitation of chasemedia. Note that β-cut APPs (lanes 5,6) runs slightly below α-cut APPs(lanes 3,4), as expected. Lanes 7,8: antibody C7 immunoprecipitation ofcell lysates. Lanes 9,10: antibody 1282 immunoprecipitation of celllysates.

FIGS. 4A-4B. Differential inhibition of Aβ(42) and Aβ(40) formation. (A)Labeled K695sw cells were chased with the indicated concentrations ofMDL 28170, and the chase media were precipitated with 21F12 (upperpanel) followed by antibody 1282 (lower panel). (B) Quantitation of theeffect of 200 μM MDL 28170 on Aβ and p3 in the chase media byphosphorimaging. The bars show the relative pixel number compared to anuntreated control (set at 100%). The decreases in Aβ_(total) andp3_(total) relative to an untreated control were significant (*)(two-tailed t-test, n=4, p<0.001), whereas the decreases in Aβ(42) andp3(42) upon treatment with MDL 28170 did not reach significance. Thedifference in reduction of Aβ(42) level vs. total Aβ level issignificant (two-tailed t-test, n=4, p<0.01). The difference ininhibition of p3(42) vs. total p3 is also significant (two-tailedt-test, n=4, p<0.05).

FIGS. 5A-5F. Differential inhibition of Aβ production by K695sw cellsunder a variety of conditions. (A) treatment with 1 μM PDBu (P)decreases both Aβ(42) and Aβ_(total). Upper panel: antibody 21F12precipitation, lower panel: subsequent antibody 1282 precipitation.(B-E) K695sw cells were chased with (M) or without (0) 200 μM MDL 28170,and the chase media were immunoprecipitated (B) first with antibody BC05(upper panel) and then with antibody 1282 (lower panel); (C) first withantibody C42 (upper panel) and then with antibody 1282 (lower panel);(D) first with antibody 2G3 (upper panel) and then with antibody 21F12(lower panel); (E) after splitting the medium into two aliquots, eitherwith antibody 21F12 (upper panel) or antibody 1282 (lower panel). (F)cells were labeled for 3 h in the presence of 100 μM MDL 28170, and themedium was immunoprecipitated first with antibody 21F12 (upper panel)and then with antibody 1282 (lower panel).

FIGS. 6A-6C. Differential inhibition of Aβ production in different celltypes. 35S-methionine labeled cells were chased with the indicatedconcentrations of MDL 28170 and precipitated with antibody 21F12 (upperpanel) followed by antibody 1282 (lower panel). (A) V717F cells. Therelatively low APP expression leads to a faint Aβ42 band. (B) CHO695cells. Note that in CHO cells the p3 brands migrate as doublets, asdescribed (Koo and Squazzo, (1994) “Evidence that production and releaseof amyloid β-protein involves the endocytic pathway.” J. Biol. Chem.269:17386-17389). (C) SKN695 cells. While Aβ(42) and p3(42) are slightlyincreased at 200 μM MDL 28170, Aβ_(total) and p3_(total) are decreased.

FIG. 7. Shows a summary sketch of some of the known proteolyticprocessing pathways of APP.

FIG. 8A. Shows a schematic of a combination cDNA/genomic coding sequenceallowing alternative splicing of the KPI and OX-2 exons.

FIG. 8B. Shows a schematic of a combination cDNA/genomic coding sequencebearing, a mutation at position 717 and allowing alternative splicing ofthe KPI and OX-2 exons.

FIG. 9. Diagram of the intermediate constructs used to construct the APPsplicing cassette and the PDAPP vector.

FIGS. 10A-H. The DNA sequence (SEQ ID NO: 2) and the deduced amino acidsequence (SEQ ID NO: 3) of APP770.

FIG. 11. Diagram, of the genomic region of APP present in the PDAPPconstruct. The sizes of original introns 6, 7 and 8, as well as thesizes of the final introns are indicated in the diagram. The locationsof the deletions in introns 6 and 8 present in the PDAPP construct alsoare indicated.

FIG. 12. Schematic map of the PDAPP vector, a combination cDNA/genomicAPP construct.

FIGS. 13A-13O depict the percent inhibition of Aβ by several compoundsusing the brain cell culture method described in Example VIII. Thecharts show inhibition of total Aβ (“% Aβ inhibition”), inhibition ofAβ(42) (“% 1-42 inhibition”) and inhibition of the metabolism of MTT (“%MTT inhibition”) (greater inhibition indicates greater cytotoxicity).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “binding substance” refers to a polypeptide substantiallyencoded by an immunoglobulin gene or immunoglobulin genes, or fragmentsthereof, which specifically bind and recognize an analyte (antigen). Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Antibodies exist, e.g., as intactimmunoglobulins or as a number of well characterized fragments producedby digestion with various peptidases. This includes, e.g., Fab′ andF(ab)′₂ fragments. The term “binding substance,” as used herein, alsoincludes antibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies.

The term “immunoassay” is an assay that utilizes a binding substance tospecifically bind an analyte. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the analyte.

A binding substance “specifically binds to” or “is specificallyimmunoreactive with” a protein when the binding substance functions in abinding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedbinding substances bind preferentially to a particular protein and donot bind in a significant amount to other proteins present in thesample. Specific binding to a protein under such conditions requires anantibody that is selected for its specificity for a particular protein.A variety of immunoassay formats may be used to select bindingsubstances specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York, for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens andproteins for which antisera or monoclonal antibodies are available.Binding substances can be made detectible, e.g., by incorporating aradio-label into the peptide, and used to detect antibodies specificallyreactive with the peptide. A label often generates a measurable signal,such as radioactivity, fluorescent light or enzyme activity, which canbe used to quantitate the amount of bound label.

II. In Vitro Screening

Alzheimer's disease is characterized by the initial deposition ofAβ(x-≧41) in the form of amyloid plaques in the brain. Therefore,effective treatments for AD are expected to decrease the production ofthese peptides, whereas agents that hasten progress of the disease areexpected to increase production of the peptide. Prior screening methodslooked for compounds that decreased total Aβ. However, since Aβ(x-≧41)peptides are a small fraction of total Aβ, those assays could notdetermine whether the compound specifically inhibited Aβ(x-≧41)peptides. As results described herein indicate, compounds can alter theproduction of Aβ(x-≦40) but not Aβ(x-≧41). Because Aβ(42) is the majorconstituent of neuritic plaques, it is useful to identify compounds thatspecifically inhibit the production of Aβ(x-≧41) peptides, either inaddition to, or instead of Aβ(x-≦40) peptides. Accordingly, thisinvention provides methods for screening compounds that specificallyelevate or decrease the production of the amount of Aβ(x-≧41) by a celland compounds that elevate or decrease production of both Aβ(x-≧41) andAβ(x-≦40) (e.g., total Aβ), or of one or the other of these peptides.Compounds that decrease production of Aβ(x-≧41) are candidates for usein treating the disease, while compounds that increase its productionmay hasten the disease and are to be avoided by humans.

Screening methods of this invention for determining whether a testcompound specifically alters the amount of Aβ(x-≧41) produced by a cellinvolve administering the compound to the cell, usually in culture,measuring the amount of Aβ(x-≧41) specifically produced by the cell, anddetermining whether this amount is greater than, less than or the sameas an amount the cell is expected to produce in the absence of thecompound. If the amounts are different, then the compound affects theproduction of Aβ(x-≧41) by the cell. This amount can be measured, forexample, in a sample from the culture, such as medium conditioned by thecell in culture, or in extracts derived from cells harvested from theculture.

The expected amount generally will be a control amount determined bymeasuring Aβ(x-≧41) produced by the cell in the absence of the compound.However, one also may determine the expected amount by extrapolation;measuring the amount of Aβ(x-≧41) produced upon administration ofdifferent amounts of the compound to the cell, and using these figuresto calculate the expected amount. In certain instances measuring acontrol amount for the purposes of comparison may not be necessarybecause the effect of the compound on Aβ(x-≧41) production is clear. Forexample, a compound may render Aβ(x-≧41) undetectable in a cell thatnormally produces detectable amounts, indicating that the compounddecreases Aβ(x-≧41) production from the amount expected in its absence.

In another aspect, this invention provides screening methods fordetermining whether a compound alters the production of Aβ(x-≧41) by acell to a different degree than it alters the production of total Aβ orAβ(x-≦40) by the cell. These methods are useful for determining whethera compound alters the production of Aβ(x-≧41) in addition to total Aβ,or alters the production of one or the other of Aβ(x-≧41) and Aβ(x-≦40).The methods involve administering the compound to the cell (usually inculture). Then, the degree to which the compound alters the productionof Aβ(x-≧41), specifically, by the cell is determined. The degree towhich the compound alters the production of total Aβ or Aβ(x-≦40) by thecell also is determined. Then, the degrees are compared. The comparisonindicates whether the compound alters the production of Aβ(x-≧41)instead of or in addition to Aβ(x-≦40).

Determining the degree to which a compound alters the production of oneor the other peptide generally involves measuring the specific amount ofthe peptide in a sample from the culture; and comparing it with theamounts expected in a sample from a culture comprising the cell to whichno compound has been administered.

III. Amyloid-β Peptide and Related Proteins and Peptides

Various cellular processing pathways for APP are presented in FIG. 7.The terms “amyloid-β peptide,” “Aβ” or “βAP” as used herein refer to anapproximately 4.2 kD protein which, in the brains of AD, Down'sSyndrome, HCHWA-D and some normal aged subjects, forms the subunit ofthe amyloid filaments comprising the senile (amyloid) plaques and theamyloid deposits in small cerebral and meningeal blood vessels (amyloidangiopathy). Aβ can occur in a filamentous polymeric form (in this form,it exhibits the Congo-red and thioflavin-S dye-binding characteristicsof amyloid described in connection therewith). Aβ can also occur in anon-filamentous form (“preamyloid” or “amorphous” or “diffuse” deposits)in brain tissue, in which form no birefringent staining by Congo redoccurs. A portion of this protein in the insoluble form obtained frommeningeal blood vessels is described in U.S. Pat. No. 4,666,829.

Aβ is an approximately 39-43 amino acid fragment of a largemembrane-spanning glycoprotein, referred to as the β-amyloid precursorprotein (APP), encoded by a gene on the long arm of human chromosome 21.Forms of Aβ longer than 43 amino acids are also contemplated herein. Aβis further characterized by its relative mobility in SDS-polyacrylamidegel electrophoresis or in high performance liquid chromatography (HPLC).A sequence for a 43-amino acid-version of Aβ is:

(SEQ ID NO:1) 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr 11 Glu Val HisHis Gln Lys Leu Val Phe Phe 21 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala31 Ile Ile Gly Leu Met Val Gly Gly Val Val 41 Ile Ala Thr.

As used herein, Aβ also refers to related polymorphic forms of Aβ,including those that result from mutations in the Aβ region of the APPgene.

The term “Aβ fragment” as used herein refers to fragments anddegradation products of Aβ which are generated at low concentrations bymammalian cells. Particular Aβ fragments have a molecular weight ofapproximately 3 kD and are presently believed to include peptides with,for example, amino acid residues 3-34, 6-27, 6-34, 6-35, 6-42, 11-34,11-40, 11-43, 12-43, 17-40 and 17-42 of Aβ (Vigo-Pelfrey et al. (1993)J. Neurochem. 61:1965-1968).

As used herein, the term “Aβ(x-≧41)” refers to Aβ or an Aβ fragmentwhose amino-terminus begins at amino acid number 1 of Aβ or thereafter(i.e., which is amino-terminally truncated), and whose carboxy-terminusextends beyond amino acid number 40. These peptides and fragmentscomprise a heterogenous group. For example, Aβ(6-42), Aβ(11-43) andAβ(12-43) all have been found in the CSF. However, this list is notmeant to be exclusive. Other peptides from among the group are presumedto exist in the culture media of cells expressing APP and are detectablewith the methods described herein. As used herein the term “Aβ(42)”refers to Aβ or an Aβ fragment whose C-terminal amino acid is # 42 ofAβ.

As used herein, the term “Aβ(x-≦40)” refers to Aβ or an Aβ fragmentwhose amino-terminus begins at amino acid number 1 of Aβ or which isamino-terminally truncated, and whose carboxy-terminus extends nofurther than amino acid number 40. These peptides and fragments alsocomprise a heterogenous group. The term “Aβ(40)” refers to Aβ or an Aβfragment whose C-terminal amino acid is #40 of Aβ.

As used herein, the term “p3” refers to a peptide whose amino acidsequence is substantially similar to Aβ, but whose amino-terminal aminoacid begins at amino acid 17 of Aβ. The term “p3 fragment” as usedherein refers to fragments and degradation products of p3. Whereas p3 isproduced through a different processing pathway than Aβ, for thepurposes of the detection methods of this invention, p3 and p3 fragmentsare considered to be a subset of Aβ peptides, because certain detectiontechniques that recognize Aβ solely from the carboxy-terminus generallyalso will recognize p3. Also it is shown that the same apparentmechanisms generate the p3 and Aβ carboxy-termini.

The term “Aβ junction region” as used herein refers to a region of Aβwhich is centered at the site between amino acid residues 16 and 17(Lys¹⁶ and Leu¹⁷), which is a principal target for proteolyticprocessing of APP. Such processing, referred to as “α-secretory”processing, results in a variety of APP fragments which may, forexample, terminate at amino acid 16 of Aβ and which, therefore, arepotentially immunologically cross-reactive with antibodies to the intactAβ molecule which are to be used in the methods of the presentinvention. Antibodies raised against a synthetic peptide including aminoacid residues 13-28 have been found to display the requisite specificityfor the junction region.

The term “amyloid-β precursor protein” (APP) as used herein is definedas a polypeptide that is encoded by a gene of the same name localized inhumans on the long arm of chromosome 21 and that includes the Aβ regionwithin the carboxyl third of its coding region. APP is a glycosylated,single-membrane-spanning protein expressed in a wide variety of cells inmany mammalian tissues. Examples of specific isotypes of APP which arecurrently known to exist in humans are the 695-amino acid polypeptidedescribed by Kang et al. (1987) Nature 325:733-736; the 751-amino acidpolypeptide described by Ponte et al. (1988) Nature 331:525-527 (1988)and Tanzi et al. (1988) Nature 331:528-530; and the 770-amino acidpolypeptide described by Kitaguchi et al. (1988) Nature 331:530-532.Examples of specific variants of APP include point mutations which candiffer in both position and resultant neuropathological phenotype (forreview of known variant mutations see Hardy (1992) Nature Genet.1:233-234).

The term “Aβ-related condition” as used herein is defined as includingAlzheimer's disease (which includes familial Alzheimer's disease),Down's Syndrome, HCHWA-D, and advanced aging of the brain.

IV. Cells Expressing Aβ(x-≧41)

Because it is a therapeutic goal to discover compounds that inhibit theproduction of Aβ(x-≧41), the test cells used in the methods of thisinvention generally are ones that are able to secrete Aβ(x-≧41).

In vitro monitoring of Aβ(x-≧41) levels in conditioned medium from asuitable cell culture may be used for drug screening. By growing cellsunder conditions which result in the secretion of Aβ(x-≧41) into theculture medium, and exposing the cells to test compounds, the effect ofthese test compounds on Aβ(x-≧41) secretion can be observed.

Suitable cell lines include human and animal cell lines, such as,preferably, primary human neurons, and primary neurons from transgenicmice harboring human APP genes, e.g., cells from a transgenic PDAPPanimal (e.g., mouse), as well as a 293 human kidney cell line, a humanneuroglioma cell line, a human HeLa cell line, a primary endothelialcell line (e.g., HUVEC cells), a primary human fibroblast line or aprimary lymphoblast line (including endogenous cells derived frompatients with APP mutations), a primary human mixed brain cell culture(including neurons, astrocytes and neuroglia), or a Chinese hamsterovary (CHO) cell line. Particularly useful are cells stably transfectedwith APP₆₉₅ carrying the mutation V717I (valine to isoleucine atposition 717 in the APP770 numbering system). Cell lines whichpreferentially increase the levels or ratios of Aβ(x-≧41) would beparticularly useful in the methods of invention. Useful mutants atposition 717 (the Hardy mutation) include V717F, V717I or V717G.

Preferred for use in drug screening methods according to the presentinvention are cell lines capable of expressing APP variants whichoverproduce Aβ. By “overproduce,” it is meant that the amount of Aβproduced from the variant APP will be at least about one-and-a-halftimes and preferably at least two or five times greater than the amountproduced from any or all of the normal APP isoforms, e.g., the 695, 751,and 770 amino acid isoforms which have been previously described.Particularly preferred are APP variants having one or several amino acidsubstitutions directly amino-terminal of the Aβ cleavage site. Forexample, K293 cells which express an APP₆₉₅ DNA bearing a doublemutation (Lys⁵⁹⁵->Asn⁵⁹⁵ and Met⁵⁹⁶->Leu⁵⁹⁶ (695 numbering system))found in a Swedish FAD family produce approximately five-to-eight-foldmore Aβ than cells expressing normal APP (Citron et al. (1992) Nature,360:672-674). The mutation at residue 596 appears to be principallyresponsible for the increase.

V. Expression Vectors for APP

Host cells transfected with a recombinant expression vector that encodesAPP also are useful as cells in the screening methods of this invention.A plasmid that carries sequences encoding APP is pCMV695 (Selkoe et al.(1988) Proc. Natl. Acad. Sci USA 85:7341-7345).

Nucleic acids encoding APP can be obtained by methods known in the art.For example, a nucleic acid encoding an APP can be isolated bypolymerase chain reaction of cDNA or genomic DNA from a human brain cDNAlibrary or a human genomic library using primers based on the DNAsequence of APP. PCR methods are described in, for example, U.S. Pat.No. 4,683,195; Mullis et al. (1987) Cold Spring Harbor Symp. Quant.Biol. 51:263; and Erlich, ed., PCR Technology, (Stockton Press, NY,1989).

Mutant versions of APP, such as the Swedish mutation, can be made bysite-specific mutagenesis of other nucleic acids encoding APP, or byrandom mutagenesis caused by increasing the error rate of PCR of theoriginal polynucleotide with 0.1 mM MnC1 ₂ and unbalanced nucleotideconcentrations.

The construction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques alsowell known in the art. Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,(Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc.).

Nucleic acids used to transfect cells with sequences coding forexpression of the polypeptide of interest generally will be in the formof an expression vector that includes expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used herein, the term nucleotide sequence “coding forexpression of” a polypeptide refers to a sequence that, upontranscription and translation of mRNA, produces the polypeptide. As anyperson skilled in the art recognizes, this includes all degeneratenucleic acid sequences encoding the same amino acid sequence. This caninclude sequences containing, e.g., introns. As used herein, the term“expression control sequences” refers to nucleic acid sequences thatregulate the expression of a nucleic acid sequence to which it isoperatively linked. Expression control sequences are “operativelylinked” to a nucleic acid sequence when the expression control sequencescontrol and regulate the transcription and, as appropriate, translationof the nucleic acid sequence. Thus, expression control sequences caninclude appropriate promoters, enhancers, transcription terminators, astart codon (i.e., ATG) in front of a protein-encoding gene, splicingsignals for introns, maintenance of the correct reading frame of thatgene to permit proper translation of the mRNA, and stop codons.

The recombinant nucleic acid can be incorporated into an expressionvector comprising expression control sequences operatively linked to therecombinant nucleic acid. The expression vector can be adapted forfunction in prokaryotes or eukaryotes by inclusion of appropriatepromoters, replication sequences, markers, etc.

The expression vector can be transfected into a host cell for expressionof the recombinant nucleic acid. Host cells can be selected for highlevels of expression in order to purify the protein. The host cell canbe a prokaryotic or eukaryotic cell selected to study the activity of anenzyme produced by the cell. The cell can be, e.g., a cultured cell or acell in vivo.

Transfected host cells useful in this invention include human kidney 293cell lines such as K695sw, and K695⁷¹⁷¹, glioma cell lines such asHS695, and neuroblastoma cell lines such as SKN695, described in theExperimental section.

VI. Measuring Aβ(x-≧41)

Aβ peptides can be detected by any method known in the art. Preferably,the method involves an immunoassay employing binding substances specificfor the peptides. Optionally, one can detect Aβ peptides by determiningtheir size, e.g., by HPLC or by mass spectrometry.

A. Binding Substances

One step of the screening methods of this invention involves measuringthe amount of at least one Aβ(x-≧41) peptide, specifically, in a sample.Measuring Aβ(x-≧41) peptides specifically means measuring Aβ(x-≧41)peptides so as to distinguish that molecule from shorter species of Aβ,i.e., those species whose carboxy-terminus extends no further than aminoacid # 40 of Aβ.

Specific measurement of Aβ(x-≧41) preferably is performed by the use ofbinding substances that specifically recognize Aβ(x-≧41) peptides, e.g.,binding substances that recognize amino acids of Aβ beyond amino acid #40.

Another method of this invention involves screening compounds todetermine their ability to alter the production of both Aβ(x-≧41)peptides and total Aβ or Aβ(x-≦40) peptides. Such methods can involvethe use of binding substances that can distinguish Aβ(x-≦40) peptidesfrom longer species of Aβ, such as Aβ(x-≧41) peptides.

B. Immunoassays

The use of immunological detection techniques, i.e., immunoassaysemploying binding substances, is preferred. Particularly suitabledetection techniques include ELISA, Western blotting, radioimmunoassay,and the like. Suitable. immunological methods employing a singleantibody are also contemplated, for example, radioimmunoassay using anantibody specific for ≧41 forms of Aβ, or single antibody ELISA methods.It will be clear that the particular forms of Aβ detected by suchmethods depend upon the particular binding substances employed. Forexample, binding substances directed to the junction region may detectAβ(x-≧41) peptides whose amino termini do not extend to amino acid no. 1of Aβ. Also, binding substances directed to the carboxy-terminal end ofAβ(x-≧41) may detect peptides ending at amino acids 41, 42 or 43.Therefore, determining the specificity of the binding substances willassist in determining exactly which Aβ(x-≧41) peptides the assay isdetecting.

In one embodiment, the method to detect Aβ(x-≧41) peptides is animmunoassay involving two antibodies. One antibody is specific for anepitope containing amino acids beyond number 40 in Aβ, and anotherantibody is capable of distinguishing Aβ and Aβ fragments from other APPfragments which might be found in the sample. In particular, it has beenfound that antibodies which are monospecific for the junction region ofAβ are capable of distinguishing Aβ from other APP fragments. Thejunction region of Aβ is centered at amino acid residues 16 and 17,typically spanning amino acid residues ˜13 to ˜28. Such“junction-recognizing” antibodies may be prepared using syntheticpeptides having that sequence as an immunogen.

A preferred immunoassay technique is a two-site or “sandwich” assay.This assay involves a capture binding substance, usually bound to asolid phase, and a labelled detection binding substance. In this method,Aβ(x-≧41) peptides are captured from the sample using a first bindingsubstance specific for Aβ(x-≧41) peptides (usually bound to a solidphase). The capture of Aβ(x-≧41) peptides is detected using a labeledsecond binding substance specific for Aβ. Labeled binding substancesinclude, for example, those directed to the junction region (amino acids˜13 to ˜28) or binding substances specific for amino-terminal aminoacids (1-5 or 1-12).

Particular methods for preparing such antibodies and utilizing suchantibodies in an exemplary ELISA are set forth in the Experimentalsection hereinafter and in related U.S. patent application Ser. No.07/965,972, supra. A sandwich assay using an antibody against thejunction region can be used to specifically measure Aβ and Aβ fragmentswhose amino-terminus begins before amino acid 13 of Aβ. Such assays donot recognize p3 or p3 fragments, since those peptides begin at aminoacid # 17 of Aβ.

Antibodies specific for Aβ(x-≧41), i.e., which do not cross react withAβ(≦40), are particularly useful in the methods of this invention. Theseantibodies can be made by immunizing animals with synthetic peptidesthat include amino acids beyond number 40 of Aβ. For example, thesynthetic peptide can include amino acids 33-42. A specific example ofthe production of such an antibody is provided in the Experimentalsection.

The particular peptides measured from among the group of all Aβ(x-≧41)depends on the particular measuring method used. In the case of usingbinding substances, such as antibodies, the binding substance can bedirected to one or more from among the group of peptides. For example,an antibody raised against amino acids 33-42 of Aβ that does not crossreact with Aβ(1-40) will bind to Aβ(x-42). It also may bind to Aβ(x-41)and Aβ(x-43). According to one embodiment of, the invention, the methodinvolves determining the amount of Aβ(x-≧41) having at least amino acids13-41 of Aβ. These species can be measured using a sandwich assayemploying antibodies that recognize the junction region (amino acids13-26) and antibodies produced by immunization with a hapten having Aβamino acids 33-42. Total Aβ can be measured using a capture antibody tothe junction region (e.g., the 266 antibody, described herein) and areporter antibody that should detect virtually all the Aβ peptides andAβ fragments, e.g., an antibody raised against amino acids 1-12 of Aβ.

C. Pulse-Chase Assays

Another method of measuring the amount of Aβ(x-≧41) in a sample involvespulse-chase procedures. In these methods, the culture is pulsed with aradioactive label for protein, e.g., a radioactive amino acid such as³⁵S methionine. Then the culture is chased without the label. Then thecompound is administered to the cells. Then the cells are contacted witha binding substance specific for Aβ(x-≧41) and the amount of radioactivelabel attached to the binding substance is determined.

D. Preparation of Antibodies

Antibodies specific for Aβ can be prepared, e.g., by immunizing ananimal with a peptide whose amino acid sequence corresponds with aminoacids ˜13 to ˜28 of Aβ. Antibodies specific for Aβ(x-≧41) can beprepared, e.g., by immunizing an animal with a peptide whose amino acidsequence corresponds with amino acids 33-42 of Aβ. Antibodies specificfor Aβ(x-≦40) can be prepared, e.g., by immunizing an animal with apeptide whose amino acid sequence corresponds with amino acids 33-40 or28-40 of Aβ. Antibodies against the junction region are useful fordetecting total Aβ.

Synthetic polypeptide haptens may be produced by the well-knownMerrifield solid-phase synthesis technique in which amino acids aresequentially added to a growing chain (Merrifield (1963) J. Am. Chem.Soc. 85:2149-2156). Suitable peptide haptens will usually comprise atleast five contiguous residues within Aβ and may include more than sixresidues. The amino acid sequences may be based on the sequence of Aβset forth above.

Once a sufficient quantity of polypeptide hapten has been obtained, itmay be conjugated to a suitable immunogenic carrier, such as serumalbumin, keyhole limpet hemocyanin, or other suitable protein carriers,as generally described in Hudson and Hay, Practical Immunology,Blackwell Scientific Publications, Oxford, Chapter 1.3, 1980. Anexemplary immunogenic carrier utilized in the examples provided below isα-CD3ε antibody (Boehringer-Mannheim, Clone No. 145-2C11).

Antibodies specific for the desired epitope may be produced by in vitroor in vivo techniques. In vitro techniques involve exposure oflymphocytes to the immunogens, while in vivo techniques require theinjection of the immunogens into a suitable vertebrate host. Suitablevertebrate hosts are non-human, including mice, rats, rabbits, sheep,goats, and the like. Immunogens are injected into the animal accordingto a predetermined schedule, and the animals are periodically bled, withsuccessive bleeds having improved titer and specificity. The injectionsmay be made intramuscularly, intraperitoneally, subcutaneously, or thelike, and an adjuvant, such as incomplete Freund's adjuvant, may beemployed.

If desired, monoclonal antibodies can be obtained by preparingimmortalized cell lines capable of producing antibodies having desiredspecificity. Such immortalized cell lines may be produced in a varietyof ways. Conveniently, a small vertebrate, such as a mouse, ishyperimmunized with the desired immunogen by the method just described.The vertebrate is then killed, usually several days after the finalimmunization, the spleen cells removed, and the spleen cellsimmortalized. The manner of immortalization is not critical. Presently,the most common technique is fusion with a myeloma cell fusion partner,as first described by Kohler and Milstein (1975) Nature 256:495-497.Other techniques including EBV transformation, transformation with bareDNA, e.g., oncogenes, retroviruses, etc., or any other method whichprovides for stable maintenance of the cell line and production ofmonoclonal antibodies. Specific techniques for preparing monoclonalantibodies are described in Antibodies: A Laboratory Manual, Harlow andLane, eds., Cold Spring Harbor Laboratory, 1988.

In addition to monoclonal antibodies and polyclonal antibodies(antisera), the detection techniques of the present invention will alsobe able to use antibody fragments, such as F(ab), Fv, V_(L), V_(H), andother fragments. In the use of polyclonal antibodies, however, it may benecessary to adsorb the anti-sera against the target epitopes in orderto produce a monospecific antibody population. It will also be possibleto employ recombinantly produced antibodies (immunoglobulins) andvariations thereof as now well described in the patent and scientificliterature. See, for example, EPO 8430268.0; EPO 85102665.8; EPO85305604.2; PCT/GB 85/00392; EPO 85115311.4; PCT/US86/002269; andJapanese application 85239543, the disclosures of which are incorporatedherein by reference. It would also be possible to prepare otherrecombinant proteins which would mimic the binding specificity ofantibodies prepared as just described.

VII. Kits

This invention also provides kits for performing assays of theinvention. The kits include means for detecting specifically Aβ(x-≧41)and means for detecting specifically Aβ(x-≦40). The means can includeany means known or described above, e.g., binding substances.

In one embodiment, the kit includes a binding substance specific forAβ(x-≧41) (i.e., that does not cross react with Aβ(≦40)), and a bindingsubstance specific for Aβ(≦40) (i.e., that does not cross react withAβ(x-≧41)). Such kits are useful, e.g., in pulse-chase assays.

In another embodiment, the kit is useful for immunoassays including twoantibodies for each antigen. For example, the kit can further comprise abinding substance specific for the junction region of Aβ. Suchantibodies are useful for the capture or detection of both Aβ(x-≧41) andAβ(≦40). In one kit useful for a sandwich ELISA, the binding substancespecific for the junction region is bound to a solid phase, and thebinding substances specific for Aβ(x-≧41) and Aβ(≦40) are detectablylabeled.

The detectable labels can be any known and used in the art including,e.g., a biotinylation label, a radioactive label, a light scatteringlabel, an enzymatic label, a fluorescent label and the like. When thelabel is enzymatic, the kit can further comprise a substrate for theenzyme.

VIII. Test Compounds

The test compounds can be any molecule, compound, or other substancewhich can be added to the cell culture or administered to the testanimal without substantially interfering with cell or animal viability.Suitable test compounds may be small molecules (i.e., molecules whosemolecular mass is no more than 1000 Daltons), biological polymers, suchas polypeptides, polysaccharides, polynucleotides, and the like. Thetest compounds will typically be administered to the culture medium at aconcentration in the range from about 1 nM to 1 mM, usually from about10 μM to 1 mM. The test compounds will typically be administered at adosage of from 1 ng/kg to 100 mg/kg, usually from 10 μg/kg to 1 mg/kg.

Test compounds which are able to inhibit secretion or production ofAβ(x-≧41) are considered as candidates for further determinations of theability to block β-amyloid production in animals and humans. Suchcompounds can be tested in in vivo studies, as described below.Inhibition of secretion or production indicates that cleavage of Aβbetween amino acids 42/43 has likely been at least partly blocked,reducing the amount of Aβ(x-≧41) available for forming β-amyloidplaques.

IX. In Vivo Screening

Animal models currently are being used to study Alzheimer's disease.(See, e.g., International Patent Application WO 93/14200, U.S. patentapplication Ser. No. 08/143,697, filed Oct. 27, 1993, now U.S. Pat. No.5,604,102, U.S. Pat. No. 5,387,742, and U.S. application Ser. No.08/486,538, filed Jun. 7, 1995.) These models are useful for screeningcompounds that alter the production of Aβ(x-≧41) in the assays of thisinvention for their ability to affect the course of Alzheimer's disease,both to ameliorate and aggravate the condition. Transgenic mammalianmodels, more particularly, rodent models and in particular murine,hamster and guinea pig models, are suitable for this use.

A preferred non-human transgenic animal is one whose cells harbor aPDAPP construct. A PDAPP construct is a nucleic acid construct thatcomprises a mammalian promoter operatively linked to a cDNA-genomic DNAhybrid coding for the expression of APP. The cDNA-genomic DNA hybridcontains a cDNA sequence encoding APP770 or a cDNA sequence encodingAPP770 with a naturally occurring mutation (e.g., a Hardy mutation orthe Swedish mutation) substituted with genomic DNA sequences. Thegenomic DNA sequences consist of exon 6 and an amount of the adjacentdownstream intron sufficient for splicing, the KI and OX-2 coding regionand an amount of each of their upstream and downstream intronssufficient for splicing, and exon 9 and an amount of the adjacentupstream intron sufficient for splicing, substituted into thecorresponding region of the cDNA sequence encoding APP770, or the cDNAencoding APP770 with a naturally occurring mutation. The construct istranscribed and differentially spliced in mammalian cells to form mRNAmolecules that encode and that are translated into APP695, APP751 andAPP770. In certain embodiments, the construct contains a PDGF-β promoteroperatively linked with a hybrid sequence encoding an APP gene harboringa Hardy mutation (V717F), and the SV40 polyadenylation signal. Oneversion of the PDAPP construct is presented in Example IX.

Another useful non-human animal model harbors a copy of an expressibletransgene sequence which encodes the Swedish mutation of APP(asparagine⁵⁹⁵-leucine⁵⁹⁶). The sequence generally is expressed in cellswhich normally express the naturally-occurring endogenous APP gene (ifpresent). Such transgenes typically comprise a Swedish mutation APPexpression cassette, in which a linked promoter and, preferably, anenhancer drive expression of structural sequences encoding aheterologous APP polypeptide comprising the Swedish mutation. Aβ levelscan be measured in any body fluid or tissue sample, for example, brainhomogenate.

The transgenic animals that are usually produced by introducing thetransgene or targeting construct into a fertilized egg or embryonic stem(ES) cell, typically by microinjection, electroporation, lipofection, orbiolistics. The transgenic animals express the Swedish mutation APP geneof the transgene (or homologously recombined targeting construct),typically in brain tissue. Preferably, one or both endogenous APPalleles is inactivated and incapable of expressing the wild-type APP.

In all cases, it will be necessary to obtain a control value which ischaracteristic of the level of Aβ(x-≧41) and/or total Aβ or Aβ(x-≦40)production in the test animal in the absence of test compound(s). Incases where the animal is sacrificed, it will be necessary to base thevalue on an average or a typical value from other test animals whichhave been transgenically modified to express the Swedish mutant of humanAPP but which have not received the administration of any test compoundsor any other substances expected to affect the level of production ofAβ(x-≧41) and/or total Aβ or Aβ(x-≦40). Once such control level isdetermined, test compounds can be administered to additional testanimals, in which deviation from the average control value wouldindicate that the test compound had an effect on the γ-secretaseactivity in the animal. Test substances which are considered positive,i.e., likely to be beneficial in the treatment of Alzheimer's disease orother β-amyloid-related conditions, will be those which are able toreduce the level of Aβ(x-≧41) production, preferably by at least 20%,more preferably by at least 50%, and most preferably by at least 80%.

The following examples are offered by way of illustration, not by way oflimitation.

EXPERIMENTAL

I. Antibody Preparation

A. Monoclonal Antibodies to the Aβ Junction Region

Monoclonal antibodies to the junction region of Aβ were prepared using asynthetic peptide spanning amino acid residues 13-30, except that AI,amino acids 30 and 31, were substituted with GC (the “junctionpeptide”). The junction peptide was conjugated to an immunogen (α-CD3εantibody; Clone No. 145-2C11, Boehringer-Mannheim) usingm-maleimidobenzoyl-N-hydroxysuccinimide ester (MHS) according to themanufacturer's (Pierce) instructions.

A/J mice were immunized initially intraperitoneally (IP) with the Aβconjugate mixed with complete Freund's adjuvant. Fourteen days later,the mice were boosted IP with the Aβ conjugate mixed with phosphatebuffered saline (PBS) at 14 day intervals. After six total boosts, themice were finally boosted intravenously with Aβ conjugate mixed withincomplete Freund's adjuvant and fused 3 days later. Fusion of spleencells with P3.653 myeloma cells was performed as described in Oi andHerzenberg, Selective Methods in Cellular Immunology, Mishell andShigii, Eds., W. H. Freeman and Company, San Francisco, Chapter 17(1980). Serum titers and initial screens were performed by the RIAmethod described below. Several clones were expanded to a 24-well plateand subjected to further analysis as described below. Clones of interestwere produced in mouse ascites.

The RIA method used to screen serum bleeds and fusion hybridomasupernatants was based upon a method developed by Wang et al. (1977) J.Immunol. Methods 18:157-164. Briefly, the supernatant (or serum) wasincubated overnight at room temperature on a rotator with ¹²⁵I-labeledAβ₁₋₂₈ and Sepharose® 4B beads to which sheep anti-mouse IgG had beencoupled via cyanogen bromide. The beads from each well were harvestedonto glass fiber filter discs with a cell harvester and washed severaltimes with PBS. The filter discs were then transferred to gamma tubesand the bound radioactivity was counted in a gamma counter.

All hybridomas were tested for binding to Aβ₁₋₂₈ using the methoddescribed above in the initial screen, and then retested 3 days later.Aβ₁₋₂₈ positive clones were further characterized for reactivity to¹²⁵I-labeled Aβ₁₋₁₆ using the RIA method described above. No clones werefound to bind Aβ₁₋₁₆. In a peptide capture ELISA, all clones were foundto react with Aβ₁₃₋₂₈ while no clones reacted with Aβ₁₇₋₂₈. Therefore,it was determined that all clones had an epitope within the junctionregion spanning amino acids 16 and 17.

Based on results of the above assays, several clones were expanded into24 well plates. These clones were further characterized by saturationanalysis. Supernatants at the 50% titer point (as determined by the RIAmethod described above) were added to wells containing Sepharose®-sheepanti-mouse IgG beads, a constant amount of ¹²⁵I-labeled Aβ₁₋₂₈, andvarying amounts of unlabeled junction peptide or Aβ₁₇₋₂₈. Theconcentration of cold peptide for 50% inhibition was determined for eachantibody. For the Aβ₁₇₋₂₈, no inhibition was seen at 100 ng/well for anyclones. The 50% inhibition point for junction peptide ranged from 10-80ng/well. The clones were also characterized based on reactivity inWestern blots. Based on titer point, sensitivity (as determined by the50% inhibition point), and reactivity on Western blot, several cloneswere produced in ascites. Antibodies from hybridoma designated 266 (the“266 antibody”) was selected for use as a capture antibody in the assaysdescribed below.

Supernatants from wells containing hybridoma cells were screened forantibody with the ability to capture ¹²⁵I labeled Aβ 1-42 in solution byimmunoprecipitation (Notebook 1101).

B. 2G3 Production

Antibody 2G3, specific for Aβ(x-40), was produced by injecting femaleA/J mice intraperitoneally with 100 μg immunogen per injection. Theimmunogen consisted of the peptideNH₂-Cys-NH—CH₂—(CH₂)₅—CO-GLMVGGVV-COOH (SEQ ID NO: 5), coupled to sheepanti-mouse IgG using maleimidohexanoyl-N-hydroxysuccinimide. Theimmunogen was emulsified with Freund's complete adjuvant for the firstimmunization, and all subsequent immunizations were with 100 μg ofimmunogen emulsified with Freund's incomplete adjuvant at approximatelytwo week intervals.

Three days before fusion, a mouse was boosted with PBS solutionscontaining 50 μg immunogen intravenously and 50 μg intraperitoneally inPBS. The mouse was sacrificed, the spleen was removed, splenocytes wereisolated and fused with the SP2/0 mouse myeloma using a modification ofthe method of Koehler and Milstein.

Supernatants from wells containing hybridoma cells were screened for theability to produce antibody which recognizes Aβ(1-40) which had beencoated onto an ELISA plate. “Positives” were further screened for theirability to capture ¹²⁵I Aβ(1-40) in solution by immunoprecipitation.

C. Production of 21F12

Antibody 21F12, specific for Aβ(x≧41) was produced by immunizing A/Jmice intraperitoneally with 100 μg of immunogen per injection. Theimmunogen consists of the synthetic peptideNH₂-Cys-NH—CH₂(CH₂)₅—CO-GLMVGGVVIA-COOH (SEQ ID NO: 4) coupled to sheepanti-mouse IgG using maleimidohexanoyl-N-hydroxysuccinimide (MHS). Theimmunogen was emulsified with Freund's complete adjuvant for the firstimmunization, and all subsequent immunizations were with 100 μg ofimmunogen emulsified with Freund's incomplete adjuvant at approximatelytwo week intervals.

Three days before the fusion, a mouse was injected with 50 μg ofimmunogen each intravenously and intraperitoneally of immunogen in PBS.Three days post injection, the spleen was removed, splenocytes wereisolated and fused with SP2/0 following a modification of the method ofKoehler and Milstein.

D. Specificity of 2G3 and 21F12

The specificities of antibody 2G3 and 21F12 are demonstrated in FIGS. 1and 2. In this assay, antibody 2G3 or 21F12 was coated into the wells ofan ELISA plate by diluting the purified antibody to a concentration of10 μg/ml in Well-coating Buffer (0.01 M PO₄ pH 8.5), and pipetting 100μl of the antibody solution into each well. The solution was leftovernight at room temperature and then was removed by aspiration. Thenon-specific sites of the well were blocked by the addition of 200 μl0.25% Human Serum albumin in PBS and incubated for at least one hour atroom temperature. The blocking solution was removed and the wells werewashed one time with wash buffer (Tris buffered Saline, 0.05% Tween 20).

Standards containing between 80-20,000 pg/ml of either Aβ(1-40) orAβ(1-42) were then prepared by dilution in Specimen Diluent (1 mM PO₄,0.15 M NaCl, pH 7.4, 0.6% Bovine Serum albumin, globulin-free, 0.05%Triton X-405 and 0.05% Thimerosal), and 100 μl of each of thesestandards were added to the appropriate wells. The standards wereincubated for one hour at room temperature, then aspirated and the wellswashed four times with wash buffer.

100 μl of a second antibody (the reporter antibody) was added at aconcentration of 0.5 μg/ml in specimen diluent. This reporter antibodyis biotinylated 3D6 (which recognizes Aβ (1-5)) prepared by the reactionof antibody with NHS-biotin (Pierce). This was allowed to incubate onehour at room temperature, and then washed four times with wash buffer.

To each well, 100 μl of a 1/5000 dilution of avidin HRP (Vector Labs)was added and allowed to incubate one hour at room temperature. Thewells were washed four times in wash buffer, and 100 μl of Slow TMB(Pierce) were added to each well and incubated fifteen minutes. Thereaction was stopped by the addition of 25 μl of 2 M H₂SO₄ and theplates were read at 450-650 on a Vmax reader (Molecular Devices)(Notebook 1344).

As can be seen in FIG. 1, antibody 2G3 reacts strongly with Aβ(1-40),but has essentially no cross-reactivity with Aβ(1-42). In FIG. 2, it isshown that antibody 21F12 similarly has very high specificity, in thiscase for Aβ(1-42) over Aβ(1-40). At a concentration of 20,000 pg/ml lessthan 0.4% of cross reactivity is observed.

II. ELISA Assay

A. Binding of Capture Antibody to Microtiter Wells

A monoclonal antibody against Aβ(x≧41) or Aβ(x≦40) is diluted to aconcentration of 10 μg/ml in a buffer containing 0.23 g/L NaH₂PO₄.H₂O,26.2 g/L Na₂HPO₄.7H₂O, 1 g/L NaN₃, pH 8.5. One hundred μl/well of thissolution is then dispensed in a 96 well white Dynatech Microlite 2, 96well flat-bottomed plate. The plates are sealed and incubated overnightat room temperature. Following coating, the remaining solution wasaspirated and the non-specific binding sites are blocked with 200 μL perwell of (NaH₂PO₄.H₂O) 0.2 g/L, Na₂HPO₄.7H₂O 0.8 g/L, human serum albumin(HSA) crystallized and lyophilized 2.5 g/L, pH 7.4. These plates areblocked by incubating for 1 hour at room temperature in the blockingsolution.

B. Assay Protocol

The calibrators are prepared from a stock solution of Aβ₁₋₄₂, 1 μg/ml,in DMSO. In specimen diluent ((NaH₂PO₄.H₂O) 0.2 g/L, Na₂HPO₄.7H₂O 2.16g/L, NaN₃ 0.5 g/L, bovine serum albumin (BSA) (globulin free) 6 g/L,triton x-405 0.5 ml/L NaCl 8.5 g/L, pH 7.4.), the highest calibrator,1000 pg/ml (10 μl Aβ₁₋₄₂ stock (1 μg/ml DMSO) in 10 ml casein specimendiluent) is prepared. Sequential dilutions are made in specimen diluentto obtain 500, 250, 125, 62.5 and 31.25 pg/ml concentrations of Aβ₁₋₄₂.

One hundred μL per well calibrators or samples are applied to themicrotiter plates. The plates are sealed and incubated for 1 hour atroom temperature. The plates are then washed three times with washingbuffer (NaCl 80 g/L, KCl 3.85 g/L, Tris-HCl 31.75 g/L, tween-20 0.5ml/L, pH 7.5).

Antibody is diluted in specimen diluent to 1 μg/ml and 100 μl is addedper well. The plate is covered and incubated for 1 hour at roomtemperature. The plate is washed three times with washing buffer. Thealkaline phosphatase affinity purified F(ab′)2 fragment donkeyanti-rabbit IgG (H+L) (Jackson) is diluted 1:1000 in specimen diluent.One hundred μl/well is added. The plate is covered and incubated for 1hour at room temperature. The plate is washed three times with washingbuffer, then 100 μl/well of chemiluminescent substrate is added. Thechemiluminescent substrate is prepared by diluting the chemiluminescentreagent, AMPPD (Tropix), and an enhancer, emerald green (Tropix), 1:1000and 1:100 respectively in 1M diethanolemine buffer, pH 10, containing 1mM MgCl₂ and 0.2% NaN₃. The plates are sealed and incubated for 10 to 15minutes at room temperature. Solution is not aspirated. This time mayhave to be optimized for different antibody lots.

Chemiluminescence is read and expressed as relative chemiluminescenceunits (CLU) after 15 minutes using a Dynatech ML 1000.

III. Inhibition of APP Processing by the MDL 28170 Inhibitor

We first set out to reproduce the published results of Higaki et al.(1995) Neuron, 14:651-659 on the action of the compound MDL 28170 on APPprocessing, using human kidney 293 cells stably expressing APP₆₉₅ withthe Swedish FAD mutation (K695sw cells). Our experiments were done usinga pulse-chase paradigm: K695sw cells were labeled for 2 hours with35S-methionine and then chased for 2 hours in the presence of 200 μM MDL28170. Aliquots of the chase media from treated and untreated cells weresubjected to SDS-PAGE. No significant differences in the amounts of themajor secreted cellular proteins were detected (FIG. 3, lanes 1, 2),suggesting that under the conditions of the experiment, MDL 28170 doesnot interfere with general protein secretion.

We next analyzed the chase media for changes in the amounts of α- andβ-cleaved APP_(s), using antibodies specific for each form. Antibody1736 specifically immunoprecipitates α-cleaved APP_(s) (Haass et al.(1994) J. Biol. Chem. 269:17741-17748; Haass et al. (1995) Nature Med.1:1291-1296). This antibody revealed an increase in α-APP_(s) productionupon treatment (FIG. 3, lanes 3, 4), indicating that MDL 28170 does notsignificantly inhibit α-secretase.

192sw specifically immunoprecipitates the β-cleaved APP_(s) speciesending with the Swedish mutant met₅₉₆ (Knops et al. (1995) J. Biol.Chem. 270:2419-2422; Haass et al. (1995) Nature Med. 1:1291-1296).Immunoprecipitation with this antibody showed that MDL 28170 does notsignificantly inhibit β-secretase activity (FIG. 3, lanes 5, 6).

We next analyzed lysates of the K695sw cells for changes in cellularfull-length APP and its C-terminal fragments using antibody C7, directedto the last 20 amino acids of APP (Podlisny et al. (1991) Am. J. Pathol.138:1423-1435). This antibody precipitates N′- and N′/O′-glycosylatedfull-length APP and its 10 kDa C-terminal fragment (residues 613-695 ofAPP₆₉₅) that remains membrane bound after α-secretase cleavage. Upontreatment with MDL 28170, a striking increase in the level of the 10 kDaC-terminal fragment was observed (FIG. 3, lanes 7, 8).

In K695sw cells, the 12 kDa C-terminal fragment (residues 597-695 ofAPP₆₉₅) which remains membrane bound after β-secretase cleavage cannotbe easily resolved and detected by antibody C7 (Citron et al., 1995). Wetherefore precipitated this fragment using antibody 1282 raised tosynthetic Aβ(1-40). This antibody can precipitate the 12 kDa C-terminalbut not the 10 kDa fragment, and therefore the faint 12 kDa band is notovershadowed by the much more abundant 10 kDa band. Whereas no 12 kDafragment was detectable in untreated cells, this band was clearlyobserved upon treatment with MDL 28170 (FIG. 3, lanes 9, 10).

In summary, no significant inhibition of α- or β-secretase cleavage, butan increase in both the 10 and 12 kDa C-terminal fragments was observedupon treatment with MDL 28170, strongly supporting the role of thiscompound as a γ-secretase inhibitor which inhibits the conversion of the10 and 12 kDa fragments to the p3 and Aβ peptides, respectively.

IV. MDL 28170 Inhibits the Production of Aβ(40) and p3(40) but notAβ(42) and p3(42)

MDL 28170 had previously been shown to inhibit the secretion of Aβ andp3 and therefore had been suggested to inhibit γ-secretase. Thisinhibition was observed by immunoprecipitating media from treated cellswith a polyclonal antibody raised to synthetic Aβ(1-40) (Higaki et al.,(1995) Neuron, 14:651-659). Since the vast majority of secreted Aβ andp3 peptides end at amino acid 40, this experiment does not distinguishwhether only the major γ-secretase cleavage at position 40 is inhibited.

To address this question, we performed pulse-chase experiments on K695swcells using different doses of MDL 28170, followed by sequentialimmunoprecipitation of the same media first with 21F12, a monoclonalantibody raised against amino acids 33-42 of Aβ (described above) whichspecifically precipitates Aβ peptides ending at position 42, and thenwith antibody 1282 which precipitates all forms of Aβ and p3 (Haass etal., (1992b) Nature 359:322-325) (FIG. 4A). Interestingly, 21F12precipitated not only Aβ, but also p3 peptides, thus demonstrating theexistence of secreted p3(42), which had not been described before. TotalAβ and total p3 were strongly and significantly decreased with doses ofMDL 28170>50 μM (e.g., at 200 μM p<0.001). In contrast, Aβ(42) andp3(42) showed a bell-shaped dose response curve, with only a small andinsignificant decrease at 200 μM, the dose used by Higaki et al. (1995)Neuron, 14:651-65.9, and the highest dose tested here. Using MDL 28170at 200 μM, the experiment was repeated four times and the results werequantitated by phosphorimaging (FIG. 4B). These data indicate that underthe conditions described above the differential effect is significantfor both Aβ and p3.

V. Differential Inhibition is Accomplished Under a Number of Conditions

To make sure that the differential effect observed in Aβ(40)/Aβ(42) andp3(40)/p3(42) precipitations is meaningful, we performed a number ofcontrol experiments using the K695sw cells. First we treated K695swcells in a 2 h pulse 2 h chase paradigm with 1 μM of the phorbolesterPDBu, which has been shown to decrease total Aβ but increase total p3,probably by diverting βAPP substrate from the β-secretase pathway to theα-secretase route (Buxbaum et al., (1993) “Protein phosphorylationinhibits production of alzheimer amyloid β/A4 peptide,” Proc. Natl.Acad. Sci. USA 90:9195-9198; Hung et al., (1993) “Activation of proteinkinase C inhibits cellular production of the amyloid β-protein,” J.Biol. Chem. 268:22959-22962). This effect should be independent of thesubsequent γ-secretase cleavage and thus the 40 and 42 forms of eachmetabolite should be equally decreased or increased if theimmunoprecipitation paradigm used here works correctly. Indeed, whenconditioned media of PDBu-treated cells was precipitated with 21F12, theexpected decrease in Aβ(42) and increase in p3(42) were observedindicating that Aβ(42) immunoprecipitation signal does reflect changesin the amounts of precipitable material. Subsequent immunoprecipitationwith R1282 shows the same effect for total Aβ and total p3 (FIG. 5A).

The statement that Aβ(42) and p3(42) are not decreased by MDL 28170depends critically on the quality of the 21F12 antibody. To confirm theeffects observed with this antibody, two other previously publishedAβ(42)-specific antibodies were used in the pulse chase paradigm withMDL 28170 at 200 μM. The monoclonal antibody BC05 has been extensivelyused in ELISA assays to detect Aβ(42) (Asami-Odaka et al., (1995)Biochemistry 34:10272-10278; Gravina et al., (1995) J. Biol. Chem.270:7013-7016; Suzuki et al., (1994) Science 264:1336-1340).

When media from MDL 28170-treated K695sw cells was precipitated withthis antibody, we observed an actual increase in both Aβ(42) and p3(42).The subsequent precipitation with R1282 showed the usual decrease intotal Aβ and p3 (FIG. 5B).

The polyclonal antibody C42 has also been shown to be specific forAβ(42) (Saido et al., (1994) Spatial resolution of the primaryβ-amyloidogenic process induced in postischemic hippocampus. J. Biol.Chem. 269:15253-15257). Likewise, this antibody did not show a decreasein Aβ(42) and p3(42) upon treatment whereas the subsequent precipitationwith R1282 showed the usual decrease in total Aβ and p3 (FIG. 5C).

The decrease in Aβ(40) and p3(40) was also found when the monoclonalantibody, 2G3 (described above) specific for the free carboxyl-terminusof Aβ(40) and p3(40) was used to precipitate first, followed by 21F12(FIG. 5D). As expected, the differential inhibition of Aβ production byMDL 28170 was also detected when the precipitations were carried out notsequentially (as described above) but in parallel after the standardpulse-chase. That is, aliquots of media from treated cells and fromuntreated cells were precipitated with 21F12 for the Aβ42 forms andother aliquots were precipitated with antibody 1282 for total Aβ andtotal p3. This parallel precipitation produced the same result as thesequential precipitations described above (FIG. 5E).

In summary, three different Aβ(42) end-specific antibodies show that MDL28170 does not strongly decrease Aβ(42) and p3(42) production whereas amonoclonal antibody to Aβ(40) and p3(40) and different polyclonalantibodies to total Aβ and p3 show a strong decrease. Finally,immunoprecipitation with 21F12 followed by antibody 1282 again revealedthis differential inhibition when the inhibitor (100 μM) was appliedduring a 3 h labeling period instead of in a pulse-chase format (FIG.5F).

VI. Differential Inhibition is Observed in Several Cell Lines

To check whether the differential inhibition is specific for K695sw,three additional cell lines were treated in the standard 2 h pulse-2 hchase paradigm, and the conditioned media were precipitated first with21F12 and then with antibody 1282. The kidney cell line K695_(717I)expresses APP₆₉₅ carrying the 717I mutation. This line was chosenbecause it produces increased levels of Aβ(42) due to the mutation(Suzuki et al., (1994) Science 264:1336-1340). At 200 μM MDL 28170, nodecrease of Aβ(42) and p3(42) was observed, whereas Aβ(40) and p3(40)were substantially reduced. (FIG. 6A). The Chinese Hamster ovary cellline CHO695 transfected with wild-type βAPP₆₉₅ cDNA was treated with 200μM MDL 28170 and only a slight decrease of Aβ(42) and p3(42) wasobserved, whereas Aβ(40) and p3(40) were substantially reduced. (FIG.6B). The human neuroblastoma cell-line SKN695 expressing wild typeβAPP₆₉₅ was treated with 200 μM MDL 28170 (FIG. 6C). While Aβ(42) andp3(42) were slightly increased, total Aβ and total p3 were stronglydecreased. Thus, differential inhibition of Aβ(42) vs Aβ(40) and p3(42)vs p3(40) production is not only observed in K695sw but also in a cellline with an Alzheimer's disease linked βAPP717 missense mutation, in ahamster cell line and in a human neural cell line expressing wild typeβAPP.

VII. Experimental Procedures

A. Cell Lines

All transfected cell lines described here carry derivatives of pCMV695,a plasmid carrying APP₆₉₅ under control of the CMV promoter (Selkoe,(1988) Proc. Natl. Acad. Sci. 85:7341-7345) K695sw are human embryonickidney 293 cells stably transfected with a construct carrying theAD-linked double (“Swedish”) mutation K595N/M596L (Citron et al. (1992)Nature, 360:672-674); K695_(717I) are 293 cells stably transfected withAPP₆₉₅ carrying the mutation V717I (valine to isoleucine at position 717in the APP770 numbering system). CHO695 are Chinese hamster ovary cells(CHO) stably transfected with pCMV695 (Oltersdorf (1990) J. Biol. Chem.265:4432-4437) SKN695 are SK-N-SH human neuroblastoma cells stablytransfected with pCMV695.

B. Pulse-Chase Experiments and Immunoprecipitations

To analyze the effect of MDL 28170 on the processing of APP, cells weregrown to confluence in two 10 cm dishes, pulse-labeled with 600 μCi of[³⁵S]-methionine in 4 ml of serum-free medium for 2 hours and thenchased for 2 hours with 4 ml medium containing 10% fetal bovine serumand the indicated final concentration of MDL 28170 (initially dissolvedat 200 mM. in DMSO). Control dishes were treated with DMSO alone.

Conditioned media and cell lysates were analyzed by immunoprecipitation,as described (Haass, et al. (1992) Nature 359:322-325). Polyclonalantibody R1736 to residues 595-611 of APP₆₉₅ was used to precipitateα-APP_(s) (Haass, (1994) J. Biol. Chem. 269:17741-17748). This antibodyrecognizes an epitope that is specific for the free COOH-terminus ofα-cleaved APP_(s). Polyclonal antibody R1282 was generated to syntheticAβ1-40 (Haass, et al. (1992) Nature 359:322-325). This antibodyprecipitates total Aβ and p3 (and small, variable amounts of APP_(s))from the media of cultured cells (Haass, et al. (1992) Nature359:322-325). The monoclonal antibody 2G3 was raised to peptideC(Aminoheptanoic acid)GLMVGGVV [(SEQ ID NO: 5) and specificallyprecipitates Aβ(40) and p3(40). Twenty μg of this antibody were used toimmunoprecipitate the chase media of 2 dishes. The monoclonal antibody21F12 was raised to peptide C(Aminoheptanoic acid)GLMVGGVVIA (SEQ ID NO:4) and specifically precipitates Aβ(42) and p3(42). Twenty μg of thisantibody were used to immunoprecipitate the chase media of 2 dishes. Themonoclonal antibody BC05 specifically detects Aβ(42) and p3(42) (Suzukiet al., (1994) Science 264:1336-1340). The polyclonal antibody C7against the last 20 residues of the APP cytoplasmic tail (Podlisny,(1991) Am. J. Pathol. 138:1423-1435) precipitates N′- and N⇑ plusO⇑-glycosylated full-length APP as well as its C-terminal proteolyticfragments. The antibody sw192 (Knops et al. (1995) J. Biol. Chem.270:2419-2422; Haass et al. (1995) Nature Med.1:1291-1296) specificallyprecipitates β-cleaved APP_(s) carrying the Swedish mutation.

SDS-PAGE of immunoprecipitates of cell extracts or of Aβ from media wascarried out on 10-20% Tris-Tricine gels (Novex), whereas APP_(s)precipitates were electrophoresed on 10% SDS-polyacrylamide Tris Glycinegels. All quantitations were performed with a Phosphorimager 400A usingImage-QuaNT software (Molecular Dynamics). It should be noted thepulse/chase-immunoprecipitation method allows one to assess any changesin Aβ and p3 simultaneously in the same assay, with each of thesepeptides being visualized directly in the electrophoretic gel.

VIII. Human Neurons

Human neurons were cultured as previously described (Seubert et al.Nature (1992) 359:325-327) except that the cells were seeded into 6-wellplates in neuronal medium without fetal bovine serum but supplementedwith B27 (Gibco). Cells were cultured for 2-3 weeks in serum free mediumprior to use.

PDAPP mouse brain cells from 16 day old fetal cerebral cortex werecultured following the protocol for human neurons except the cells wereseeded into 24-well plate clusters in neuronal medium with 50 fetalbovine serum (Sigma) and 5% Chang's supplement (Irvine Scientific).Cells were cultured for 5-7 days prior to being used in experiments.

The procedure for examining the effects of substances on Aβ productionis as follows. Fresh medium is added to the culture wells and thencollected after ˜24 (8-30) hrs. This is the “control” sample from eachwell that the treated sample will be compared to. Fresh medium,containing the substance to be tested is then added and again harvestedafter a further ˜24 hr (8-30) incubation. After collection of this“treated” sample, a cytotoxicity assay is performed on the cells. Toperform the cytotoxicity assay, cells are incubated in media containingthiazolyl blue (MTT, Sigma) at 1 mg/ml for 15 minutes. The media arethen discarded, and the precipitates are analyzed by solubilization in abuffer containing 50% DMF and 20% SDS. The solubilized dye wasquantitated on a Molecular Devices Vmax.

Control and treated samples of culture media are assayed for total Aβusing a sandwich ELISA consisting of two monoclonal antibodies. Thefirst antibody 266, specific to amino acids 13-28 of Aβ, is used as acapture antibody (Seubert et al., Nature, supra). The second antibody,3D6 which is specific to amino acids 1-5 of Aβ, was biotinylated andserved as a reporter antibody. The 3D6 biotinylation procedure employedthe manufacturer's protocol for NHS-biotin (Pierce) labeling ofimmunoglobulins, except 100 mM sodium bicarbonate, pH 8.5, buffer wasused. The 3D6 antibody does not recognize secreted APP or full-lengthAPP but does recognize Aβ species that begin at position 1.

The samples were also assayed for Aβ(42) with an Aβ(42) specificsandwich ELISA that employed the monoclonal antibody 21F12, which wasgenerated against amino acids 33-42 of Aβ, as the capture antibody. Thisantibody is specific for longer forms of Aβ since it does notcross-react with Aβ(1-40) in ELISA or competitive radioimmunoassay(RIA). Biotinylated 3D6 is also the reporter antibody in this assay.

The 266 and 21F12 mAbs were coated at 10 μg/ml into 96-well immunoassayplates (Costar) overnight at room temperature. The plates were aspiratedand blocked with 0.25% human serum albumin PBS buffer for at least 1hour at room temperature, then stored desiccated at 4° C. until use. Thesamples and standards were added to the plates and incubated at roomtemperature for 1.5 hours.

The biotinylated 3D6 was diluted to 0.5 μg/ml, and incubated in thewells for 1 hour at room temperature. The plates were washed 3 timeswith wash buffer (Tris buffered saline, 0.05% Tween 20) between eachstep of the assay. Streptavidin-alkaline phosphatase (BoehringerMannheim), diluted 1:1000, was added to the wells for the total Aβassay, and avidin-HRP (Vector) diluted 1:4000 was added to the wells forthe Aβ(42) assay. These conjugates were incubated for 1 hour at roomtemperature. For the total Aβ assay the fluorometric substrate4-methyl-umbelliferyl phosphate was added to the wells for 30 minutes,then read in a Millipore Cytofluor 2350 fluorometer. The calorimetricsubstrate, Slow TMB-ELISA (Pierce), was added for the Aβ(42) assay andallowed to react for 15 minutes, after which the enzymatic reaction wasstopped with 2N H₂SO₄. The plates were read on a Molecular Devices Vmax.

Percent inhibition for both total Aβ and Aβ(42) is defined as:(1−((treated/control_(t))/(untreated/control_(u))))×100%,where treated=value from treated cells

-   -   control_(t)=value from the same treated well for the 24 hr        period prior to testing    -   untreated=value from well which received no test substance    -   control_(u)=value from the same untreated well for the 24 hr        prior to testing

It is optimal to divide the values of the treated and untreated samplesby their respective values for the 24 hrs prior to the 24 hr testperiod, as the act of changing the media alone can effect a 5-10%reduction in Aβ production (comparing the untreated to control_(u)).

Several compounds were screened for their ability to inhibit both totalAβ and Aβ(42). Results are shown in FIGS. 13A-13O.

IX. PDAPP Construct

A cDNA/genomic APP construct containing introns 6, 7 and 8 is preparedby combining APP cDNA encoding exons 1-6 and 9-18 with genomic APPsequences encoding introns 6, 7 and 8, and exons 7 and 8 (see FIGS.8A-8B). In order to create a splicing cassette small enough forconvenient insertion in a pUC vector, two deletions in intronicsequences are made. A deletion is made in intron 6 from position 143 ofintron 6 to the BamHI site located upstream of the beginning of exon 7(1658 bp before the beginning of exon 7). Another deletion is made inintron 8 from the first BamHI site in intron 8 to a site at 263 bpbefore the beginning of exon 9. To avoid confusion, these truncatedforms of APP introns 6 and 8 are referred to herein as intron Δ6 and Δ8.BamHI sites are engineered at the sites of these deletions, so that theyare marked by the presence of BamHI sites. In this construct, referredto as PDAPP, exons 7 and 8 and intron 7 are intact genomic sequences,except that the unique XhoI site in intron 7 is destroyed.

DNA fragments containing the truncated introns are generated as follows:a BamHI site is engineered 143 bp into intron 6 nucleotide byPCR-mutagenesis (“Mutagenesis by PCR” in PCR Technology: CurrentInnovations (Griffith and Griffith, eds., CRC Press, 1994) pages 69-83)and another BamHI site is engineered by PCR mutagenesis 263 bp prior tothe beginning of exon 9. These sites are engineered into separate APPgenomic DNA clones containing the junctions of exon 6 and intron 6, andintron 8 and exon 9, respectively, resulting in modified APP genomic DNAclones.

The entire cassette is assembled in the APP cDNA clone as follows (FIG.9). The 889 bp BamHI to XcmI fragment of APP cDNA containing exons 1through 5 and part of exon 6 (including nucleotides 1 to 843 of FIG. 10(SEQ ID NO: 2)) is cloned into a vector containing BamHI and XhoI sitesdownstream from the insertion site to make APP770x-oligo-x.APP770x-oligo-x is then cut with XcmI and BamHI. Then two fragments areobtained from the modified APP genomic DNA clone containing the junctionof exon 6 and intron 6 described above by cutting with XcmI and BamHI.The resulting 34 bp fragment from the XcmI in exon 6 to the XcmI inintron 6, and 131 bp fragment from the XcmI in intron 6 to theartificially created BamHI site at position 143 bp of intron 6 areligated into APP770x-oligo-x in a three-way ligation step to make APP770x-E6oligo-x. The orientation of the fragments are confirmed bysequencing. APP770x-E6oligo-x is then cut with BamHI and XhoI. Then the313 bp BamHI and XhoI fragment from the modified APP genomic DNA clonecontaining the junction of intron 8 and exon 9 is ligated intoAPP770x-E6oligo-x to make APP770xE6E9x.

APP770xE6E9x is then cut with BamHI and the 6.8 kb BamHI fragment of APPgenomic DNA encoding the KPI and OX-2 domains (exons 7 and 8) isinserted at this site. This fragment starts at the BamHI site 1658 bpupstream of the start of exon 7 and extends to the first BamHI site inintron 8. This BamHI fragment is obtained from a lambda phage genomicclone encoding this portion of the APP gene, that was obtained from aHuman Placental genomic library in the Lambda FIXII vector obtained fromStratagene. This BamHI fragment originally contained an XhoI site whichwas destroyed by cutting fill in and relegation. The locations of thedeletions are diagramed in FIG. 11. This clone, containing exons 1-8 andpart of 9, and introns 6, 7 and 8, is termed the “APP splicingcassette.” The APP splicing cassette is cut out with NruI and XhoI andused to replace the NruI to XhoI cDNA fragment of APP cDNA bearing aHardy mutation. This mutant form of APP cDNA is produced by convertingthe G at nucleotide position 2145 to T by site directed mutagenesis.This changes the encoded amino acid from Val to Phe. The resultingconstruct is a combination cDNA/genomic APP “minigene.”

Sequencing of the 6.8 kb BamHI fragment containing APP exons 7 and 8derived from the APP genomic clone used to generate this constructshowed that intron 7 is 2.6 kb long, and that the first BamHI site inintron 8, the upstream site of the deletion in intron 8 engineered intothe APP minigene construct, is 2329 bp downstream from the end of exon8. This does not coincide with the restriction map of the APP genepublished by Yoshikai et al. (1990) and Yoshikai et al. (1991).Comparison of their map to our sequence indicates that Yoshikai et al.switched the order of two EcoRI fragments in their restriction mapping.The 1.60 kb EcoRI fragment containing exon 8 is actually upstream of the1.48 kb EcoRI fragment and the 1.48 kb EcoRI fragment Yoshikai et al.mapped into intron 7 is actually in intron 8. We have confirmed thislocation for the EcoRI fragment containing exon 8 by sizing of PCRgenerated fragments from human DNA.

This APP minigene is operatively linked to the PDGF-β promoter toprovide expression of the APP cDNA/genomic construct in mammalian cells.The PDGF β-chain 5′ flanking sequence is inserted upstream of the NruIsite at the beginning of the APP minigene. This fragment includes 1.3 kbupstream of the transcription initiation site, where the PDGF-β promoterresides, and approximately 70 bp of 5′ untranslated region, ending atthe AurII site (Higgins et al. (1994)). The late SV40 polyadenylationsignal, carried on a 240 bp BamHI to BclI fragment, is added downstreamof the APP minigene. This construct, combining the PDGF-β promoter, theAPP splicing cassette, a Hardy mutation, and the SV40 polyadenylationsignal is referred to as PDAPP (FIG. 12).

The present invention provides a novel screening method for determiningwhether a compound alters the production of Aβ(x-≧41) and/or Aβ(x-≦40)or total Aβ. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Many variations of theinvention will become apparent to those of skill in the art upon reviewof this specification. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withtheir full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

1. A method for determining a compound is a compound that inhibitsproduction of Aβ(42) and Aβ(40) separately: obtaining control valueswhich are characteristic of the level of Aβ42 peptide and total Aβ orAβ40 peptide in a sample from a non-human animal used as a model ofAlzheimer's disease; administering the compound to the non-human animalused as a model of Alzheimer's disease; measuring an amount of Aβ42peptide in a sample from the non-human animal administered the compound;and measuring an amount of total Aβ or Aβ40 peptide in a sample from thenon-human animal administered the compound; wherein the measured amountsin comparison with the control values indicate differential inhibitionof Aβ (42) and Aβ (40)thus indicating the compound is a compound thatinhibits production of Aβ(42) and Aβ(40) separately.
 2. The method ofclaim 1 wherein the non-human animal is a rodent.
 3. The method of claim2 wherein the rodent is a mouse.
 4. The method of claim 1 wherein thenon-human animal is a rodent and expresses the Swedish mutation of humanbeta-amyloid precursor protein (APP).
 5. A method for determining acompound is a compound that inhibits production of Aβ (40) and not Aβ(42): obtaining control values which are characteristic of the level ofAβ42 peptide and total Aβ or Aβ40 peptide in a sample from a non-humananimal used as a model of Alzheimer's disease; administering thecompound to the non-human animal used as a model of Alzheimer's disease;measuring an amount of Aβ42 peptide in a sample from the non-humananimal administered the compound; measuring an amount of total Aβ orAβ40 peptide in a sample from the non-human animal administered thecompound; and wherein the control values in comparison with the measuredamounts indicate the compound is a compound that inhibits production ofAβ40 and not Aβ42.